APPLICANT'S ABSTRACT: Our objective is to understand the molecular basis of aldehyde dehydrogenase (ALDH) function. Liver ALDHs act in human alcohol metabolism, to clear ethanol-derived acetaldehyde. The soluble class 3 enzyme, present in nonhepatic tissues, also clears toxic products of lipid peroxidation which, in turn, are elevated by chronic ethanol intake. Toward this goal, we have now determined the first three-dimensional structure of an ALDH the class 3 rat enzyme, with a current resolution of 2.6A. The a-carbon chain has been traced in the electron density map and sidechains have been fitted. Each monomer has two distinct domains. NAD is identified bound in an open a/b structure characteristic of NAD-binding domains, but which differs in certain details from the "Rossmann folds" of other dehydrogenases. Residue conservations indicate that this is characteristic of all ALDHS. A deep channel (for aldehyde binding) contains the catalytic cysteine and other conserved residues. In the funding period, work will focus on refinement of the binary complex (E-NAD) structure and analysis of the cyclopropanone ternary complex crystals, to better understand the active site geometry. We will also model the active site cavities of the present structure, using residues from class I and 2 ALDHS, according to our multiple alignment. We are also poised to continue site-directed mutagenesis from a fresh perspective. With the structure and alignment, we now have the abillty to select residues which will be far more likely to alter, but not abolish activity. Implications of this work for human health are the obvious - that our data should help solve the structure and understand the function of ALDHs more directly involved in ethanol metabolism. Also, with this structure we have solved a structure that is relevant to tumor biology by virtue of class 3 ALDH activity on cyclophospharnide metabolites. We have also solved a structure of immediate relevance to the now-demonstrated enzymatic defect responsibleible for Sjogren-Larsson Syndrome.